Introduction
Buffalo is an integral part of agriculture in Asian countries. In India, buffaloes contribute immensely in the form of milk, meat and draft power and are considered a reliable ‘living bank’ to serve the immediate needs of rural people, who constitute the vast majority of the total population in the country. However, buffalo reproduction has certain inherent challenges such as late sexual maturity, seasonal reproductive pattern, anoestrus, silent oestrus, and a long intercalving interval (Nanda et al., Reference Nanda, Brar and Prabhakar2003; Das and Khan, Reference Das and Khan2010; Phogat et al., Reference Phogat, Pandey and Singh2016). Ovarian cysts are an important cause of infertility in the species, leading to extended calving intervals and great economic losses (Khan et al., Reference Khan, Das, Pande, Pathak and Sarkar2011; Teshome et al., Reference Teshome, Kebede, Abdela and Ahmed2016). There are several definitions used to describe ovarian follicular cysts, and the traditionally accepted definition is that they are ‘follicular structures of 2.5 cm or larger that persist for a variable period in the absence of a corpus luteum’ (Youngquist and Threlfall, Reference Youngquist and Threlfall2007). A 2003 study by Hatler et al. (Reference Hatler, Hayes, Laranja da Fonseca and Silvia2003) pointed out that follicles typically ovulate at 17 mm in diameter, so follicles that persist at that diameter or greater may be considered to be ‘cystic’. Similarly, Vanholder et al. (Reference Vanholder, Opsomer and de Kruif2006) suggested that cystic ovarian follicles (COF) should be defined as ‘follicles with a diameter of at least 2 cm that are present on one or both ovaries in the absence of any luteal tissue and that clearly interfere with normal ovarian cyclicity’.
The incidence of ovarian cysts in dairy cattle may vary from 2.7% to 15.1% (Cattaneo et al., Reference Cattaneo, Signorini, Bertoli, Bartolomé, Gareis, Díaz, Bó and Ortega2014) or from 6% to 30% (Garverick, Reference Garverick1997) with peak incidences between the interval of 14 and 40 days postpartum (López-Gatius, Reference López-Gatius2003; Yimer et al., Reference Yimer, Haron and Yusoff2018). The incidence of follicular cysts varies between 2.8% (buffalo heifers) to 4.2% (buffalo cows). This condition has been reported to be unilateral in most cases, involving mostly the right ovary (60.7%) compared with the left ovary (39.3%; Luktuke and Arora, Reference Luktuke and Arora1972). Various studies have pointed out that the basic reason for cyst formation is a failure of the preovulatory luteinizing hormone (LH) surge to occur at the appropriate time in follicular maturation (Whitlock et al., Reference Whitlock, Daniel, Wilborn, Maxwell, Steele and Sartin2011; Yeo and Colledge, Reference Yeo and Colledge2018). Follicular fluid plays a major role in autocrine and paracrine regulation and also in the physiological, biochemical and metabolic aspects of nuclear and cytoplasmic maturation of the oocyte and the process of ovulation (Hafez et al., Reference Hafez, Jainudeen, Rosnina, Hafez and Hafez2000). Because of its intimate contact with the oocyte and granulosa cells, follicular fluid can serve as a good index for the functional status of ovarian follicles (Da Broi et al., Reference Da Broi, Giorgi, Wang, Keefe, Albertini and Navarro2018; BorŞ and BorŞ, Reference BorŞ and BorŞ2020). Changes in the biochemical composition of follicular fluid may influence steroidogenesis, oocyte maturation and quality, ovulation, and transport of the oocyte to the oviduct, and preparation of the follicle for subsequent corpus luteum formation and function (Da Broi et al., Reference Da Broi, Giorgi, Wang, Keefe, Albertini and Navarro2018). Therefore, the follicular fluid composition of follicular cysts may provide valuable insight into the pathogenesis of the cystic ovarian disease. The objective of the present study was to examine changes in the follicular fluid’s biochemical, hormonal, and mineral profiles in cystic follicles of water buffalo.
Materials and methods
In total, 215 buffalo reproductive tracts, along with intact ovaries, were collected randomly from a local abattoir during the non-breeding months (May to September) and transported to the laboratory on ice within 30 min of collection. Follicles exceeding a diameter of 20 mm, present in ovaries lacking a corpus luteum, were designated as follicular cysts (Khan et al., Reference Khan, Das, Pande, Pathak and Sarkar2011). Follicular fluid was aspirated from eight cystic follicles collected from eight buffaloes and stored at −20ºC pending analysis. For comparison, follicular fluid samples from 10 preovulatory follicles (12–15 mm in diameter, oestrogen-active, collected during the follicular stage), each from a separate animal, were used.
Biochemical, hormonal and mineral analysis
Ascorbic acid estimation
Ascorbic acid was estimated following the method of Zannoni et al. (Reference Zannoni, Lynch, Goldstein and Sato1974). This method is based on the reduction of ferric iron by ascorbic acid followed by the formation of a complex of the ferrous iron product and α,α′-dipyridyl. Follicular fluid (60 µl) was mixed with 7.2 µl of 40% trichloroacetic acid (TCA) and allowed to stand on ice for 10 min, followed by centrifugation at 10,000 g for 15 min. The acidified supernatant was aspirated using a micropipette, and 20 µl was transferred to wells of a microtest plate. This was followed by sequential addition of 10 µl of 85% orthophosphoric acid, 80 µl of 1% α,α′-dipyridyl and 10 µl of 3% ferric chloride, with thorough mixing after the addition of each reagent. The plate was allowed to stand at room temperature for 15 min, and absorbance was measured using a microplate reader at 525 nm. The concentration of ascorbic acid in the samples was determined from a standard curve drawn using known standards and using GraphPad Prism software version 3.0.
Estimation of follicular fluid hormones
Progesterone (P4) was assayed using an commercial diagnostic ELISA kit (Labserv Diagnostics Ltd, UK) following the manufacturer’s instructions. The intra-assay and interassay coefficients of variance were 4% and 9.3%, respectively, and the minimum sensitivity was 0.05 ng/ml.
Estradiol and insulin were assayed in follicular fluid samples by radioimmunoassay (RIA) using a commercial RIA kit Beckman Coulter (Immunotech, France). The minimum sensitivity of the assay for E2 was 6 pg/ml and the intra-assay and interassay coefficients of variation were 12.1 and 11.2%, respectively. The minimum analytical and functional sensitivity for insulin were 0.5 µIU/ml and 1.04 µIU/ml, respectively, and the intra-assay and interassay coefficients of variation were 4.3% and 3.4%, respectively.
Determination of other biochemical constituents
Follicular fluid samples were assayed for glucose, cholesterol, total protein, alkaline phosphatase, blood urea nitrogen (BUN), creatinine, and uric acid by spectrophotometric methods using commercial diagnostic kits (Span Diagnostics India Ltd) and adopting standard procedures, as per the manufacturer’s instructions.
Follicular mineral estimation
Estimation of macrominerals (calcium, phosphorus, and magnesium)
Follicular fluid calcium (Ca), phosphorus (P), and magnesium (Mg) were estimated using commercial diagnostic kits (Span Diagnostics, India Ltd, Surat, India) adopting the procedure recommended by the manufacturer.
Estimation of microminerals (copper, cobalt, and zinc)
Copper (Cu), cobalt (Co) and zinc (Zn) were estimated in follicular fluid samples using an atomic absorption spectrophotometer (Model No. AAS 4141, Electronic Corporation of India) after acid digestion of the samples. Follicular fluid was digested following the procedure described by Kolmer et al. (Reference Kolmer, Spanbling and Robinson1951). In brief, 0.5 ml of follicular fluid in a digestion test tube was mixed with 1.5 ml of concentrated HNO3, kept at room temperature overnight, and followed by digestion on low heat (70–80ºC) using a heat bench (digestion bench) until the volume of sample was reduced to 0.5 ml. To this, 3 ml of double acid mixture (3 parts concentrated HNO3 and 1 part 70% perchloric acid) were added, and low heat digestion continued until the digested sample became clear and watery and emitted white fumes. If needed, the addition of a 3 ml double acid mixture followed by low heat digestion was repeated a couple of times. Further heating continued to reduce the volume to ∼0.5 ml. The final volume of the filtrate was made up to 5 ml with triple distilled deionized water. The final concentration was calculated by multiplying the dilution factor by the value obtained from atomic absorption spectroscopy (AAS). The characteristic wavelengths were element specific and accurate to 0.01–0.1 nm. To provide the element specific wavelengths, a light beam from a lamp whose cathode was made of the element being determined was passed through a flame (Table 1).
Table 1. Operating conditions for AAS 400 for microelements
Statistical analysis
Differences in mean concentrations of the assayed intrafollicular components between the preovulatory and cystic groups were analyzed for statistical significance using the independent sample t-test. Data are reported as mean ± standard error of the mean (SEM) unless otherwise stated.
Results
The incidence of cystic disease in this study was 3.72% (8/215), involving the right ovary in 62.5% of instances and the left ovary in 37.5% of instances. As shown in Table 2, cystic follicles showed a significantly higher concentration of progesterone and a lower concentration of estradiol and insulin compared with normal preovulatory follicles.
Table 2. Concentrations (mean ± standard error of the mean (SEM)) of hormones in follicular fluid of cystic and preovulatory follicles in water buffalo
The mean follicular fluid concentrations of different biochemical constituents and mineral profiles in follicular cysts and preovulatory follicles are presented in Table 3. Follicular cysts had a greater concentration of BUN, uric acid, creatinine, and cholesterol concentrations compared with preovulatory follicles. In contrast, ascorbic acid and glucose concentrations were less than in preovulatory follicles. Differences in total protein and alkaline phosphatase concentrations between the two groups were not significant. Follicular cysts had lower calcium and magnesium concentrations and higher copper and zinc concentrations compared with preovulatory follicles. However, the difference in phosphorus and cobalt concentrations between the two groups was not significant (Table 3).
Table 3. Concentrations (mean ± standard error of the mean (SEM)) of biochemical constituents and minerals in follicular fluid of cystic and preovulatory follicles in water buffalo
Discussion
The incidence of cystic disease was almost similar to that of a previous report by Luktuke and Arora (Reference Luktuke and Arora1972). Follicular fluid originates mainly from the peripheral plasma by transudation and secretion from the follicular cells. Its composition reflects the changes in the secretory processes of the granulosa layer and theca interna, and changes in the components of plasma due to physiological or pathological processes (Bertevello et al., Reference Bertevello, Teixeira-Gomes, Labas, Cordeiro, Blache, Papillier, Singina, Uzbekov, Maillard and Uzbekova2020). Therefore, there is a vital balance between endocrine and biochemical constituents of follicular fluid and normal follicular development (Gerard et al., Reference Gerard, Loiseau, Duchamp and Seguin2002; Da Broi et al., Reference Da Broi, Giorgi, Wang, Keefe, Albertini and Navarro2018). Changes in follicular fluid composition can be indicative of pathological conditions, including cystic follicles.
The availability of ascorbic acid in buffalo follicular fluid and its role in normal follicular development in the species has been studied previously (Meur et al., Reference Meur, Sanwal and Yadav1999). Ascorbic acid plays an essential role in steroidogenesis, follicular membrane remodelling, collagen synthesis, and antioxidant systems (Luck et al., Reference Luck, Jeyaseelan and Scholes1995; Thomas et al., Reference Thomas, Leask, Srsen, Riley, Spears and Telfer2001). In women there is sequestration of ascorbate in the follicular fluid to facilitate rapid follicular expansion during the approach to ovulation and/or post-ovulatory steroidogenesis (Aten et al., Reference Aten, Duarte and Behrman1992; Murray et al., Reference Murray, Molinek, Baker, Kojima, Smith, Hillier and Spears2001). Similar findings have been observed across bovine follicular development (Pascu et al., Reference Pascu, Suteanu and Lunca1970). A lower level of ascorbic acid in the follicular cysts might be due to a rapid increase in fluid volume, and it is possible that this lower concentration predisposes the follicle to free radical injury and impaired steroidogenesis.
Brito and Palmers (Reference Brito and Palmer2004) hypothesized various mechanisms that could potentially lead to cyst formation, although a commonly accepted hypothesis is that it results from failure of the hypothalamus to trigger the preovulatory surge of LH in response to estradiol (López-Gatius et al., Reference López-Gatius, Santolaria, Yániz, Fenech and López-Béjar2002; Whitlock et al., Reference Whitlock, Daniel, Wilborn, Maxwell, Steele and Sartin2011; Yeo and Colledge, Reference Yeo and Colledge2018). It has also been postulated that greater concentrations of P4 are possibly responsible for this hypothalamic defect (Silvia et al., Reference Silvia, Hatler, Nugent and Laranja da Fonseca2002; Robinson et al., Reference Robinson, Hunter and Mann2006). The presence of significantly higher progesterone and lower oestrogen concentrations in follicular cysts compared with preovulatory follicles in the present study supports the view that excess P4 and, subsequently, low estradiol disturb the onset of the LH surge, resulting in the persistence of follicles as follicular cysts. However, the reason for this abnormal increase in P4 and low estradiol is not yet clear and needs to be investigated in future studies.
Insulin stimulates the proliferation of follicular cells (Spicer and Stewart, Reference Spicer and Stewart1996) and oestradiol-17β production in the granulosa cells (Butler et al., Reference Butler, Pelton and Butler2004; Da Broi et al., Reference Da Broi, Giorgi, Wang, Keefe, Albertini and Navarro2018; BorŞ and BorŞ, Reference BorŞ and BorŞ2020) and is also involved in the selection of the dominant follicle towards ovulation (Fortune et al., Reference Fortune, Rivera and Yang2004; Walters et al., Reference Walters, Binnie, Campbell, Armstrong and Telfer2006). Insulin, together with increasing oestradiol levels, stimulates the dominant follicle to reach final maturation, which in turn leads to LH surge and ovulation (Kawashima et al., Reference Kawashima, Fukihara, Maeda, Kaneko, Montoya, Matsui, Shimizu, Matsunaga, Kida, Miyake, Schams and Miyamoto2007). Therefore, low insulin levels cause insufficient oestradiol-17β production. The reduced oestradiol-17β production disrupts the hypothalamic–pituitary–gonadal axis. This finally results in an aberrant LH surge and the subsequent development of a cystic follicle (Braw-Tal et al., Reference Braw-Tal, Pen and Roth2009). The findings of the current study support this notion, as low levels of insulin and oestradiol were noted in cystic follicles.
Glucose plays an important role in ovarian metabolism as it is the major energy source. A lesser concentration of glucose in the cystic follicles compared with the preovulatory follicles can be attributed to the lesser insulin concentration (Spicer and Echternkamp, Reference Spicer and Echternkamp1995) and also the active influx of the molecule in the preovulatory follicle (Landau et al., Reference Landau, Braw-Tal, Kaim, Bor and Bruckental2000) and/or the dilution due to excessive increase in follicular fluid volume. Cholesterol in follicular fluid is derived from two sources, cellular synthesis from acetate and uptake from plasma lipoprotein (Alkalby et al., Reference Alkalby, Bushra and Fahad2012). Cholesterol, present in follicular fluid, is bound to the high-density lipoprotein fraction (HDL); the low-density lipoprotein (LDL) fraction is too large to pass through the blood–follicle barrier (Bauchart, Reference Bauchart1993; Kim et al., Reference Kim, Bloom, Browne, Bell, Yucel and Fujimoto2017). The significantly higher total cholesterol concentration in cystic follicles might be attributed to the reduced conversion of cholesterol to steroid hormones, oestrogen and progesterone during steroidogenesis, and/or as size increases there may be chances of increased permeability of the cystic follicular wall because of free radical injuries and prolonged persistency of cystic follicles, as it has been reported that larger sized follicles have greater permeability compared with small-sized follicles, permitting the entrance of the larger HDL fraction (Wehrman et al., Reference Wehrman, Welsh and Williams1991; Bloom et al., Reference Bloom, Kim, Fujimoto and Browne2014).
It is well established that high-yielding cows generally suffer from NEB and are more prone to the development of cystic conditions (López-Gatius et al., Reference López-Gatius, Santolaria, Yániz, Fenech and López-Béjar2002; Hooijer et al., Reference Hooijer, van Oijen, Frankena and Noordhuizen2003). In NEB conditions, the cow’s body is conditioned for a low energy supply. Therefore, both fatty acids and amino acids are consumed and urea increases. High levels of BUN in cystic cows have also been reported by various workers (Lak, Reference Lak2007; Yousefdoost et al., Reference Yousefdoost, Samadi, Moghaddam, Hassani and Jafari2012). Lak (Reference Lak2007) suggested that high amounts of BUN in cystic cows may be related to interrupted protein metabolism. A very high correlation for urea between follicular fluid and blood serum was reported by Leroy et al. (Reference Leroy, Vanholder, Delanghe, Opsomer, Van Soom, Bols and de Kruif2004). Therefore, the increased concentration of urea nitrogen in cystic follicular fluid compared with normal preovulatory follicles possibly reflects elevated serum urea levels in the affected animals.
Creatinine is a waste product of creatine and phosphocreatine, a supplier of energy to the muscle, and is found almost exclusively (90%) in skeletal muscle tissues and formed during normal muscle contraction, and level in the blood remains unchanged from day to day. Cows with cystic ovarian disease frequently showed nymphomaniac behaviour and also, in chronic cases, the development of masculine characteristics. All these increased activities and muscle mass may lead to an increased level of creatinine in the serum of cystic cows, which might be reflected in the follicular fluid, as many serum biochemical metabolites have a very strong correlation with their follicular fluid concentration.
The observations on total protein concentration were similar to those reported earlier by various workers. The total protein content of the follicular fluid did not differ between follicle classes in dairy cows, described by Leroy et al. (Reference Leroy, Vanholder, Delanghe, Opsomer, Van Soom, Bols and de Kruif2004), in buffaloes by Arshad et al. (Reference Arshad, Ahmad, Rahman, Samad, Akhtar and Ali2005) and Abd Ellah et al. (Reference Abd Ellah, Hussein and Derar2010) and in cystic buffaloes by Khan et al. (Reference Khan, Das, Pande, Pathak and Sarkar2011). In the present study, concentrations of uric acid were significantly low in cystic follicular fluid compared with the preovulatory follicle. There was no significant difference in the concentration of alkaline phosphatase in the cystic follicle compared with the preovulatory follicle, which is similar to that reported previously by Khan et al. (Reference Khan, Das, Pande, Pathak and Sarkar2011).
Blood calcium concentration varies across the oestrous cycle, being maximum at oestrus in cattle (Burle et al., Reference Burle, Mangle, Kothekar and Kalorey1995), indicating the critical role of this ion during the follicular phase, especially in and around oestrus. Calcium content of the follicular fluid increases with the advancement of the follicle from the early follicle to the ovulatory stage, providing an optimum osmotic gradient across the follicular wall necessary for the movement of water from the blood to the antrum (Kalmath and Ravindra, Reference Kalmath and Ravindra2007). Furthermore, it was suggested that calcium is involved in the disruption of cumulus cell cohesiveness by regulating the gap junctions between the cells (Peracchia, Reference Peracchia1978). Moreover, the calcium-induced increase in the plasmin activity is believed to be a factor in weakening the follicular wall (Espey, Reference Espey1994) therefore initiating the process of ovulation. The lesser calcium concentration in cystic follicles could be one of the factors causing failure of ovulation, therefore leading to their persistence.
Release of LH–RH occurs in a Mg-dependent manner (Burrows and Barnea, Reference Burrows and Barnea1982). In an in vitro assembly, the same authors reported that Mg and ATP acted jointly to facilitate the release of LH–RH in hypothalamic granules, although Mg alone can also release LH–RH to a lower magnitude. In humans, magnesium deficiency has been reported to increase the risk of polycystic ovarian syndrome (PCOS), which was 19 times greater in Mg-deficient patients compared with those who had normal serum Mg concentrations (Sharifi et al., Reference Sharifi, Mazloomi, Hajihosseini and Mazloomzadeh2012; Hamilton et al., Reference Hamilton, Zelig, Parker and Haggag2019). Low Mg levels have been associated with anovulation. In our study a significantly low level of magnesium in cystic follicles supported the view that low levels of magnesium predispose to cyst formation in buffalo.
Classical manifestations of phosphorus deficiency on reproductive processes involve changes in oestrus and decreased ovarian activity (Ahmed, Reference Ahmed2007; Safari et al., Reference Safari, Hajian, Nasr-Esfahani, Forouzanfar and Drevet2022) characterized by anoestrus, delayed maturity (Ahmed et al., Reference Ahmed, Bashandy, Ibrahim, Shalaby, El-Moez, Ei-Moghazy and Ibrahim2010), subestrus, and irregular cycles (Hurley and Doane, Reference Hurley and Doane1989). Follicular fluid is more concentrated compared with blood in terms of phosphorus content (Tabatabaei and Mamoei, Reference Tabatabaei and Mamoei2011; Dastorani et al., Reference Dastorani, Aghadavod, Mirhosseini, Foroozanfard, Zadeh Modarres, Amiri Siavashani and Asemi2018). In the present study, lower levels of phosphorus on cystic follicles strengthened the view that phosphorus deficiency may lead to anovulation and subsequent cyst formation.
Copper concentration in cystic follicular fluid was significantly higher compared with preovulatory follicles, similarly much higher serum copper concentrations were reported in cystic cows compared with the healthy group (Yousefdoost et al., Reference Yousefdoost, Samadi, Moghaddam, Hassani and Jafari2012; Sun et al., Reference Sun, Wang, Guo, Zheng, Li, Chen and Zhang2019) leading us to speculate that this higher content in cystic follicular fluid might be influxed from higher serum concentrations or locally produced by cystic follicle metabolism; further study is warranted to confirm this.
Lower zinc levels have been reported to be associated with suboptimal steroid hormone concentrations, i.e. oestrogen and progesterone (Akhtar et al., Reference Akhtar, Farooq and Mushtaq2009; BorŞ and BorŞ, Reference BorŞ and BorŞ2020) that were attributed to the involvement of this ion in steroidogenesis (Hurley and Doane, Reference Hurley and Doane1989). Ziaee (Reference Ziaee2009) found that cystic primiparous cows had higher concentrations of zinc in serum compared with healthy multiparous cows. A higher concentration of zinc in cystic follicles could be due to the increased permeability of the blood–follicle barrier during excess follicular growth.
The concentration of cobalt was significantly high in cystic follicles compared with preovulatory follicles. This could be due to the excess permeability of the cystic follicles due to free radical injury, as it is well established that cobalt has the capacity to produce reactive oxygen species (ROS), which are highly reactive against DNA and other biomolecules (Valko et al., Reference Valko, Morris and Cronin2005).
In conclusion, from this study, significant changes in biochemical, hormonal, and mineral compositions of the follicles are associated with cystic ovarian disease in buffalo. Cystic ovarian follicles had higher follicular P4, cholesterol, urea nitrogen, creatinine, copper, zinc, and cobalt and lower E2, insulin, ascorbic acid, glucose, uric acid, calcium, magnesium and phosphorus concentrations. These changes are consistent with some of the previously proposed mechanisms underlying the development of cystic ovarian disease such as oxidative stress, aberrant endocrine function, and nutritional imbalance.
Acknowledgements
The authors are thankful to B.K. Tyagi and M.K. Pathak for their excellent technical assistance and to the Director, ICAR RC for the NEH region for placement at IVR Izatnagar as part of professional attachment training.
Funding
This research work was financially supported by the project entitled ‘Nutritional and physiological approaches for enhancing reproductive performance in cattle and buffalo’ (Reference No. 1006179) by the Indian Council of Agricultural Research, New Delhi, India.
Conflict of interest
The authors declare that there is no conflict of interest in publishing this article.
Ethical approval
The authors assert that all experimental procedures were approved by the Institute Ethical Committee of ICAR-IVRI, U.P., India.
